Human G protein-coupled receptor expressed in the dorsal root ganglia

ABSTRACT

The present invention is directed to novel G protein-coupled receptors that are found predominantly in the dorsal root ganglia. The invention encompasses both the receptor proteins as well as nucleic acids encoding the proteins. In addition, the present invention is directed to methods and compositions which utilize the receptors.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.09/254,227, filed on Mar. 3, 1999 now U.S. Pat. No. 6,696,257. The '227application represents U.S. national stage of international applicationPCT/SE98/02348, which had an international filing date of Dec. 16, 1998,and which was published in English under PCT Article 21(2) on Jul. 1,1999. The international application claims priority to Swedishapplication no. 9704836-7, filed on Dec. 22, 1997.

FIELD OF THE INVENTION

The present invention is in the general field of biological receptorsand the various uses that can be made of such receptors. Morespecifically, the invention relates to nucleic acids encoding novel Gprotein-coupled receptors and to the receptors per se.

BACKGROUND AND PRIOR ART

G protein-coupled receptors (GPCRs) constitute a family of proteinssharing a common structural organization characterized by anextracellular N-terminal end, seven hydrophobic alpha helices putativelyconstituting transmembrane domains and an intracellular C-terminaldomain. GPCRs bind a wide variety of ligands that trigger intracellularsignals through the activation of transducing G proteins (Caron, et al.,Rec. Prog. Horm. Res. 48:277–290 (1993); Freedman et al., Rec. Prog.Horm. Res. 51:319–353 (1996)).

More than 300 GPCRs have been cloned thus far and it is generallyassumed that there exist well over 1000 such receptors. Mechanistically,approximately 50–60% of all clinically relevant drugs act by modulatingthe functions of various GPCRs (Cudermann, et al., J. Mol. Med. 73:51–63(1995)). Of particular interest are receptors located in dorsal rootganglia. This region of the central nervous system is densely innervatedwith primary or afferent sensory neurons involved in the transmission,modulation and sensation of pain. Thus, receptors from this region maybe used in assays for the identification of new agents for anesthesiaand analgesia

SUMMARY OF THE INVENTION

The present invention is based upon the discovery of a novel Gprotein-coupled receptor which is distinct from previously reportedreceptors in terms of structure and in being expressed preferentially indorsal root ganglia. One dorsal root receptor (DRR) has been isolatedand sequenced from the rat and six from the human. The rat receptor wasgiven the designation rDRR-1 and its amino acid sequence is shown as SEQID NO:1. The human receptors were designated as

-   hDRR-1 (SEQ ID NO:3);-   hDRR-2 (SEQ ID NO:5);-   hDRR-3 (SEQ ID NO:7):-   hDRR-4 (SEQ ID NO:9);-   hDRR-5 (SEQ ID NO:11); and-   hDRR-6 (SEQ ID NO:13).

Unless otherwise specified, the term “DRR” as used herein refers to allof the receptors from both human and rat.

In its first aspect, the invention is directed to proteins, except asexisting in nature, comprising the amino acid sequence consistingfunctionally of a rat or human DRR as shown in SEQ ID NO:1, 3, 5, 7, 9,11, or 13. The term “consisting functionally of” is intended to includeany receptor protein whose sequence has undergone additions, deletionsor substitutions which do not substantially alter the functionalcharacteristics of the receptor. Thus, the invention encompassesproteins having exactly the same amino acid sequence as shown in thesequence listing, as well as proteins with differences that are notsubstantial as evidenced by their retaining the basic, qualitativebinding properties of the DRR receptor. The invention furtherencompasses substantially pure proteins consisting essentially of a DRRamino acid sequence, antibodies that bind specifically to a DRR (i.e.that have at least a 100 fold greater affinity for the DRR than anyother naturally occurring non-DRR protein), and antibodies made by aprocess involving the injection of pharmaceutically acceptablepreparations of such proteins into an animal capable of antibodyproduction. In a preferred embodiment, monoclonal antibody to human orrat DRR is produced by injecting a pharmaceutically acceptablepreparation of the receptor into a mouse and then fusing mouse spleencells with myeloma cells.

The invention is also directed to a substantially pure polynucleotideencoding a protein comprising the amino acid sequence consistingfunctionally of the sequence of rat DRR (as shown in SEQ ID NO:1) or ahuman DRR (as shown in SEQ ID NOs 3, 5, 7, 9, 11 or 13). This aspect ofthe invention encompasses polynucleotides encoding proteins consistingessentially of the amino acid sequences shown in the sequence listing,expression vectors comprising such polynucleotides, and host cellstransformed with such vectors. Also included are the recombinant rat andhuman DRR proteins produced by host cells made in this manner.

Preferably, the polynucleotide encoding rat DRR has the nucleotidesequence shown in SEQ ID NO:2 and the polynucleotide encoding a humanDRR has the nucleotide sequence shown in SEQ ID NO:3, 5, 7, 9, 11 or 13.It is also preferred that the vectors and host cells used for theexpression of DRR contain these particular polynucleotides.

In another aspect, the present invention is directed to a method forassaying a test compound for its ability to bind to a rat or human DRR.The method is performed by incubating a source of DRR with a ligandknown to bind to the receptor and with the test compound. The source ofthe DRR should be substantially free of other types of G protein-coupledreceptors, i.e. greater than 85% of such receptors present shouldcorrespond to the DRR. Upon completion of incubation, the ability of thetest compound to bind to the DRR is determined by the extent to whichligand binding has been displaced. The rat DRR should, preferablycorrespond to rDRR-1 as shown in SEQ ID NO:1. The human receptor shouldpreferably be hDRR-1 (SEQ ID NO:3); hDRR-2 (SEQ ID NO:5); hDRR-3 (SEQ IDNO:7); hDRR-4 (SEQ ID NO:9); hDRR-5 (SEQ ID NO:11); or hDRR-6 (SEQ IDNO:13). Either transformed cells expressing recombinant DRR may be usedin the assays or membranes can be prepared from the cells and used.Although not essential, the assay can be accompanied by thedetermination of the activation of a second messenger pathway such asthe adenyl cyclase pathway. This should help to determine whether acompound that binds to DRR is acting as an agonist or antagonist.

An alternative method for determining if a test compound is an agonistof any of the DRRs disclosed herein is to use a cell signaling assay,e.g., an assay measuring either intracellular adenyl cyclase activity orintracellular calcium concentration. The test compound is incubated withcells expressing the DRR but substantially free of other Gprotein-coupled receptors, typically a cell transfected with anexpression vector encoding the DRR. Test compounds that are agonists areidentified by their causing a statistically significant change in theresults obtained from the cell signaling assay when compared to controltransfectants not exposed to test compound. For example, the cellsexposed to the test compound may show a significant increase in adenylcyclase activity or in intracellular calcium concentration.

The invention also encompasses a method for determining if a testcompound is an antagonist of a DRR which relies upon the knownactivation of G protein-coupled receptors that occurs when suchreceptors are expressed in large amounts. This method requires that DNAencoding the receptor be incorporated into an expression vector so thatit is operably linked to a promoter and that the vector then be used totransfect an appropriate host. In order to produce sufficient receptorto result in constitutive receptor activation (i.e., activation in theabsence of natural ligand), expression systems capable of copiousprotein production are preferred, e.g., the DRR DNA may be operablylinked to a CMV promoter and expressed in COS or HEK293 cells. Aftertransfection, cells with activated receptors are selected based upontheir showing increased activity in a cell signaling assay relative tocomparable cells that have either not been transfected or that have beentransfected with a vector that is incapable of expressing functionalDRR. Typically, cells will be selected either because they show astatistically significant increase in intracellular adenyl cyclaseactivity or a statistically significant increase in intracellularcalcium concentration. The selected cells are contacted with the testcompound and the cell signaling assay is repeated to determine if thisresults in a decrease in activity relative to control cells notcontacted with the test compound. For example, a statisticallysignificant decrease in either adenyl cyclase activity or calciumconcentration relative to control cells would indicate that the testcompound is an antagonist of the DRR. Any of the DRRs disclosed hereinmay be used in these assays.

Assays for compounds interacting with a DRR may be performed byincubating a source containing the DRR but substantially free of other Gprotein-coupled receptors (e.g. a stably transformed cell) withangiotensin II or III in both the presence and absence of test compoundand measuring the modulation of intracellular calcium concentration. Asignificant increase or decrease in angiotensin-stimulated calciumdisplacement in response to test compound is indicative of aninteraction occurring at the DRR. The receptors that may be used inthese assays include rat DRR-1 and human DRR-1, DRR-2, DRR-3, DRR-4,DRR-5 and DRR-6.

In another aspect, the present invention is directed to a method forassaying a test compound for its ability to alter the expression of arat or human DRR. This method is performed by growing cells expressingthe DRR, but substantially free of other G protein-coupled receptors, inthe presence of the test compound. Cells are then collected and theexpression of the DRR is compared with expression in control cells grownunder essentially identical conditions but in the absence of the testcompound. The rat receptor is preferably rDRR-1 and the human receptormay be DRR-1; DRR-2; DRR-3; DRR-4; DRR-5; or DRR-6.

A preferred test compound is an oligonucleotide at least 15 nucleotidesin length comprising a sequence complimentary to the sequence of the DRRused in the assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Nucleotide sequence of rDRR-1: Clone 3B-32, encoding rDRR-1 wasisolated from a rat genomic library using the Promoter Finder WalkingKit (see Methods, Clontech).

The cloned gene was deposited with the international depositaryauthority Deutsche Sammlung Von Mikroorganismen Und Zellkulturen GmbH atthe address Mascheroder Weg 1 B, D-3300 Braunschweig, Germany. Thedeposit was made on Nov. 27, 1997 and was given the accession number DSM11877.

FIG. 2. Deduced amino acid sequence of DRR-1: Clone 3B-32 codes for a337 amino acid protein. The amino acid sequence begins; with the firstATG in the nucleotide sequence.

FIG. 3A–3C. Alignment of the deduced amino acid sequences of clone 3B-32(rDRR-1) with its five most homologous sequences. The boxed and shadedresidues are the ones that are identical to the rDRR-1 sequence.

FIG. 4A–4B. Amino acid alignment of the human DRR homologs: The aminoacid sequence of all 6 human homologs of rDRR-1 (hDRR-1; hDRR-2; hDRR-3;hDRR-4; hDRR-5; and hDRR-6 ) are aligned. The amino acid residuesdiffering from the clone 36 (HUMAN36.PR) are boxed. The degree ofidentity among these sequences ranges from 77% to almost 100%.

DEFINITIONS

The description that follows uses a number of terms that refer torecombinant DNA technology. In order to provide a clear and consistentunderstanding of the specification and claims, including the scope to begiven such terms, the following definitions are provided.

Cloning vector: A plasmid or phage DNA or other DNA sequence which isable to replicate autonomously in a host cell, and which ischaracterized by one or a small number of restriction endonucleaserecognition sites. A foreign DNA fragment may be spliced into the vectorat these sites in order to bring about the replication and cloning ofthe fragment. The vector may contain a marker suitable for use in theidentification of transformed cells. For example, markers may providetetracycline resistance or ampicillin resistance.

Expression vector: A vector similar to a cloning vector but which iscapable of inducing the expression of the DNA that has been cloned intoit, after transformation into a host. The cloned DNA is usually placedunder the control of (i.e., operably linked to) certain regulatorysequences such as promoters or enhancers. Promoter sequences may beconstitutive, inducible or repressible.

Substantially pure: As used herein, “substantially pure” means that thedesired product is essentially free from contaminating cellularcomponents. A “substantially pure” protein or nucleic acid willtypically comprise at least 85% of a sample, with greater percentagesbeing preferred. Contaminants may include proteins, carbohydrates orlipids. One method for determining the purity of a protein or nucleicacid is by electrophoresing a preparation in a matrix such aspolyacrylamide or agarose. Purity is evidenced by the appearance of asingle band after staining. Other methods for assessing purity includechromatography and analytical centrifugation.

Host: Any prokaryotic or eukaryotic cell that is the recipient of areplicable expression vector or cloning vector is the “host” for thatvector. The term encompasses prokaryotic or eukaryotic cells that havebeen engineered to incorporate a desired gene on its chromosome or inits genome. Examples of cells that can serve as hosts are well known inthe art, as are techniques for cellular transformation (see e.g.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. ColdSpring Harbor (1989)).

Promoter: A DNA sequence typically found in the 5 region of a gene,located proximal to the start codon. Transcription is initiated at thepromoter. If the promoter is of the inducible type, then the rate oftranscription increases in response to an inducing agent.

Complementary Nucleotide Sequence: A complementary nucleotide sequence,as used herein, refers to the sequence that would arise by normal basepairing. For example, the nucleotide sequence 5-AGAC-3 would have thecomplementary sequence 5-GTCT-3.

Expression: Expression is the process by which a polypeptide is producedfrom DNA. The process involves the transcription of the gene into mRNAand the translation of this mRNA into a polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to DRR receptor proteins, geneticsequences coding for the receptors, a method for assaying compounds forbinding to DRR receptors and a method for assaying compounds for theirability to alter DRR expression. The receptors and their nucleic acidsare defined by their structures (as shown in FIGS. 1, 2 and 4; and SEQID numbers 1–14).

It will be understood that the present invention encompasses not onlysequences identical to those shown in the figures and sequence listing,but also sequences that are essentially the same and sequences that areotherwise substantially the same and which result in a receptorretaining the basic binding characteristics of the DRR. For example, itis well known that techniques such as site-directed mutagenesis may beused to introduce variations in a protein's structure. Variations in aDRR protein introduced by this or some similar method are encompassed bythe invention provided that the resulting receptor retains the basicqualitative binding characteristics of the unaltered DRR. Thus, theinvention relates to proteins comprising amino acid sequences consistingfunctionally of the sequence of SEQ ID NO:1 (rat) and SEQ ID numbers 3,5, 7, 9, 11 and 14 (human).

I. Nucleic Acid Sequences Coding for DRR

DNA sequences coding for DRRs are expressed exclusively, or at leasthighly preferentially, in dorsal root ganglia and these ganglia mayserve as a source for the isolation of nucleic acids coding for thereceptors. In addition, cells and cell lines that express a rat or humanDRR may serve as a source for nucleic acid. These may either be culturedcells that have not undergone transformation or cell lines specificallyengineered to express recombinant DRR.

In all cases, poly A+ mRNA is isolated from the dorsal root ganglia,reverse transcribed and cloned. The cDNA library thus formed may then bescreened using probes derived from the sequences shown in theaccompanying sequence listing as SEQ ID number 2, 4, 6, 8, 10 12 or 14,depending upon the particular DRR being isolated. Probes shouldtypically be at least 14 bases in length and should be derived from aportion of the DRR sequence that is poorly conserved (see FIGS. 3 and4). Screening can also be performed using genomic libraries with one DRRgene, or a portion of the gene, serving as a probe in the isolation ofother DRR genes. For example, full length rDRR-1 may be labeled and usedto screen a human genomic library for the isolation of hDRR-1, hDRR-2etc. (see Examples section).

Alternatively genomic DNA libraries can be used to isolate DRR genes byperforming PCR amplifications with primers located at either end ofgenes (see Examples section for a description of procedures). Forexample, human genomic DNA may be amplified using the primers:

5′-GCAAGCTTTCTGAGCATGGATCCAACCGTC, and5′-CCCTCAGATCTCCAATTTGCTTCCCGACAG.

This will serve to amplify all six of the human DRR genes identifiedherein as hDRR-1; hDRR-2; hDRR-3; hDRR-4; hDRR-5; and hDRR-6. These maythen be cloned into an appropriate vector, e.g. pGEM-T (Promega), forDNA sequence analysis.

II. Antibodies to Rat and Human DRRs

The present invention is also directed to antibodies that bindspecifically to a rat or human DRR and to a process for producing suchantibodies. Antibodies that “bind specifically to a DRR” are defined asthose that have at least a one hundred fold greater affinity for the DRRthan for any other protein. The process for producing such antibodiesmay involve either injecting the DRR protein itself into an appropriateanimal or, preferably, injecting short peptides made to correspond todifferent regions of the DRR. The peptides should be at least five aminoacids in length and should be selected from regions believed to beunique to the particular DRR protein being targeted. Thus, highlyconserved transmembrane regions should generally be avoided in selectingpeptides for the generation of antibodies. Methods for making anddetecting antibodies are well known to those of skill in the art asevidenced by standard reference works such as: (Harlow et al.,Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.(1988)); Klein, Immunology: The Science of Self-Nonself Discrimination(1982); Kennett, et al., Monoclonal Antibodies and Hybridomas: A NewDimension in Biological Analyses (1980); and Campbell, “MonoclonalAntibody Technology,”in Laboratory Techniques in Biochemistry andMolecular Biology, (1984)).

“Antibody,” as used herein, is meant to include intact molecules as wellas fragments which retain their ability to bind to antigen (e.g., Faband F(ab)2 fragments). These fragments are typically produced byproteolytically cleaving intact antibodies using enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab)2 fragments). Theterm “antibody” also refers to both monoclonal antibodies and polyclonalantibodies. Polyclonal antibodies are derived from the sera of animalsimmunized with the antigen. Monoclonal antibodies can be prepared usinghybridoma technology (Kohler, et al., Nature 256:495 (1975); Hammerling,et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, M.Y., pp. 563–681 (1981)). In general, this technology involves immunizingan animal, usually a mouse, with either intact DRR or a fragment derivedfrom the DRR. The splenocytes of the immunized animals are extracted andfused with suitable myeloma cells, e.g., SP2O cells. After fusion, theresulting hybridoma cells are selectively maintained in HAT medium andthen cloned by limiting dilution (Wands; et al., Gastroenterology80:225–232 (1981)). The cells obtained through such selection are thenassayed to identify clones which secrete antibodies capable of bindingto the DRR.

The antibodies, or fragments of antibodies, of the present invention maybe used to detect the presence of DRR protein using any of a variety ofimmunoassays. For example, the antibodies may be used inradioimmunoassays or in immunometric assays, also known as “two-site” or“sandwich” assays (see Chard, T., “An Introduction to Radioimmune Assayand Related Techniques,”in Laboratory Techniques in Biochemistry andMolecular Biology, North Holland Publishing Co., N.Y. (1978)). In atypical immunometric assay, a quantity of unlabeled antibody is bound toa solid support that is insoluble in the fluid being tested, e.g.,blood, lymph, cellular extracts, etc. After the initial binding ofantigen to immobilized antibody, a quantity of detectably labeled secondantibody (which may or may not be the same as the first) is added topermit detection and/or quantitation of bound antigen (see e.g.Radioimmune Assay Method, Kirkham et al., ed., pp. 199–206, E & S.Livingstone, Edinburgh (1970)). Many variations of these types of assaysare known in the art and may be employed for the detection of the DRR.

Antibodies to a rat or human DRR may also be used in the purification ofeither the intact receptor or fragments of the receptor (see generally,Dean et al., Affinity Chromatography, A Practical Approach, IRL Press(1986)). Typically, antibody is immobilized on a chromatographic matrixsuch as Sepharose 4B. The matrix is then packed into a column and thepreparation containing the DRR desired is passed through underconditions that promote binding, e.g., under conditions of low salt. Thecolumn is then washed and bound DRR is eluted using a buffer thatpromotes dissociation from antibody, e.g., buffer having an altered pHor salt concentration. The eluted DRR may be transferred into a bufferof choice, e.g., by dialysis, and either stored or used directly.

III. Radioligand Assay for Receptor Binding

One of the main uses for DRR nucleic acids and recombinant proteins isin assays designed to identify agents capable of binding to DRRreceptors. Such agents may either be agonists, mimicking the normaleffects of receptor binding, or antagonists, inhibiting the normaleffects of receptor binding. Of particular interest is theidentification of agents which bind to the DRR and modulate adenylcyclase activity in the cells. These agents have potential therapeuticapplication as either analgesics or anesthetics. In radioligand bindingassays, a source of DRR is incubated together with a ligand known tobind to the receptor and with the compound being tested for bindingactivity. The preferred source for DRR is cells, preferably mammaliancells, transformed to recombinantly express the receptor. The cellsselected should not express a substantial amount of any other Gprotein-coupled receptors that might bind to ligand and distort results.This can easily be determined by performing binding assays on cellsderived from the same tissue or cell line as those recombinantlyexpressing DRR but which have not undergone transformation.

The assay may be performed either with intact cells or with membranesprepared from the cells (see e.g. Wang, et al., Proc. Natl. Acad. Sci.U.S.A. 90:10230–10234 (1993)). The membranes are incubated with a ligandspecific for the DRR receptor and with a preparation of the compoundbeing tested. After binding is complete, receptor is separated from thesolution containing ligand and test compound, e.g. by filtration, andthe amount of binding that has occurred is determined. Preferably, theligand used is detectably labeled with a radioisotope such as 125I.However, if desired, fluorescent or chemiluminescent labels can be usedinstead. Among the most commonly used fluorescent labeling compounds arefluorescein isothiocynate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine. Usefulchemiluminescent compounds include luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt, and oxalate ester. Any ofthese agents which can be used to produce a ligand suitable for use inthe assay.

Nonspecific binding may be determined by carrying out the bindingreaction in the presence of a large excess of unlabeled ligand. Forexamples labeled ligand may be incubated with receptor and test compoundin the presence of a thousandfold excess of unlabeled ligand.Nonspecific binding should be subtracted from total binding, i.e.binding in the absence of unlabeled ligand, to arrive at the specificbinding for each sample tested. Other steps such as washing, stirring,shaking, filtering and the like may be included in the assays asnecessary. Typically, wash steps are included after the separation ofmembrane-bound ligand from ligand remaining in solution and prior toquantitation of the amount of ligand bound, e.g., by countingradioactive isotope. The specific binding obtained in the presence oftest compound is compared with that obtained in the presence of labeledligand alone to determine the extent to which the test compound hasdisplaced receptor binding.

In performing binding assays, care must be taken to avoid artifactswhich may make it appear that a test compound is interacting with the.DRR receptor when, in fact, binding is being inhibited by some othermechanism. For example, the compound being tested should be in a bufferwhich does not itself substantially inhibit the binding of ligand to DRRand should, preferably, be tested at several different concentrations.Preparations of test compound should also be examined for proteolyticactivity and it is desirable that antiproteases be included in assays.Finally, it is highly desirable that compounds identified as displacingthe binding of ligand to DRR receptor be reexamined in a concentrationrange sufficient to perform a Scatchard analysis on the results. Thistype of analysis is well known in the art and can be used fordetermining the affinity of a test compounds for receptor (see e.g.,Ausubel, et al., Current Protocols in Molecular Biology, 11.2.1–11.2.19(1993); Laboratory Techniques and Biochemistry and Molecular Biology,Work, et al., ed., N.Y. (1978) etc.). Computer programs may be used tohelp in the analysis of results (see e.g., Munson, P., Methods Enzymol.92:543–577 (1983); McPherson, G. A., Kinetic, EBDA Ligand, Lowry—ACollection of Radioligand Binding Analysis Programs, Elsevier-Biosoft,U.K. (1985)).

The activation of receptor by the binding of ligand may be monitoredusing a number of different assays. For example, adenyl cyclase assaysmay be performed by growing cells in wells of a microtiter plate andthen incubating the various wells in the presence or absence of testcompound. cAMP may then be extracted in ethanol, lyophilized andresuspended in assay buffer. Assay of cAMP thus recovered may be carriedout using any method for determining cAMP concentration, e.g. theBiotrack cAMP Enzyme-immunoassay System (Amersham) or the Cyclic AMP[3H] Assay System (Amersham). Typically, adenyl cyclase assays will beperformed separately from binding assays, but it may also be possible toperform binding and adenyl cyclase assays on a single preparation ofcells. Other “cell signaling assays” that can be used to monitorreceptor activity are described below.

IV. Identification of DRR Agonists and Antagonists Using Cell SignalingAssays

DRRs may also be used to screen for drug candidates using cell signalingassays. To identify DRR agonists, the DNA encoding a receptor isincorporated into an expression vector and then transfected into anappropriate host. The transformed cells are then contacted with a seriesof test compounds and the effect of each is monitored. Among the assaysthat can be used are assays measuring cAMP production (see discussionabove), assays measuring the activation of reporter gene activity, orassays measuring the modulation of the binding of GTP-gamma-S.

Cell signaling assays may also be used to identify DRR antagonists. Gprotein-coupled receptors can be put in their active state even in theabsence of their cognate ligand by expressing them at very highconcentration in a heterologous system. For example, receptor may beoverexpressed using the baculovirus infection of insect Sf9 cells or aDRR gene may be operably linked to a CMV promoter and expressed in COSor HEK293 cells. In this activated constitutive state, antagonists ofthe receptor can be identified in the absence of ligand by measuring theability of a test compound to inhibit constitutive cell signalingactivity. Appropriate assays for this are, again, cAMP assays, reportergene activation assays or assays measuring the binding of GTP-gamma-S.

One preferred cell signaling assay is based upon the observation thatcells stably transfected with DRRs show a change in intracellularcalcium levels in response to incubation in the presence of angiotensinII or III (see Example 5). Thus, a procedure can be used to identify DRRagonists or antagonists that is similar to the radioreceptor assaysdiscussed above except that angiotensin II or III is used instead of alabeled ligand and calcium concentration is measured instead of boundradioactivity. The concentration of calcium in the presence of testcompound and angiotensin II or III is compared with that in the presenceof angiotensin II or III alone to determine whether the test compound isinteracting at the DRR receptor. A statistically significant increase inintracellular calcium in response to test compound indicates that thetest compound is acting as an agonist whereas a statisticallysignificant decrease in intracellular calcium indicates that it isacting as an antagonist.

V. Assay for Ability to Modulate DRR Expression

One way to either increase or decrease the biological effects of a DRRis to alter the extent to which the receptor is expressed in cells.Therefore, assays for the identification of compounds that eitherinhibit or enhance expression are of considerable interest. These assaysare carried out by growing cells expressing a DRR in the presence of atest compound and then comparing receptor expression in these cells withexpression in cells grown under essentially identical conditions but inthe absence of the test compound. As in the binding assays discussedabove, it is desirable that the cells used be substantially free ofcompeting G protein-coupled receptors. One way to quantitate receptorexpression is to fuse the DRR sequence to a sequence encoding a peptideor protein that can be readily quantitated. For example, the DRRsequence may be ligated to a sequence encoding haemaglutinin asdescribed in Example 5 and used to stably transfect cells. Afterincubation with test compound the hemagglutininn/receptor complex can beimmunoprecipitated and western blotted with anti-haemaglutinin antibody.Alternatively, Scatchard analysis of binding assays may be performedwith labeled ligand to determine receptor number. The binding assays maybe carried out as discussed above and will preferably utilize cells thathave been engineered to recombinantly express DRR.

A preferred group of test compounds for inclusion in the DRR expressionassay consists of oligonucleotides complementary to various segments ofthe DRR nucleic acid sequence. These oligonucleotides should be at least15 bases in length and should be derived from non-conserved regions ofthe receptor nucleic acid sequence. Sequences may be based upon thoseshown as SEQ ID numbers 2, 4, 6, 8, 10, 12 or 14.

Oligonucleotides which are found to reduce receptor expression may bederivatized or conjugated in order to increase their effectiveness. Forexample, nucleoside phosphorothioates may be substituted for theirnatural counterparts (see Cohen, J., Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press (1989)). The oligonucleotidesmay be delivered to a patient in vivo for the purpose of inhibiting DRRexpression. When this is done, it is preferred that the oligonucleotidebe administered in a form that enhances its uptake by cells. Forexample, the oligonucleotide may be delivered by means of a liposome orconjugated to a peptide that is ingested by cells (see e.g., U.S. Pat.Nos. 4,897,355 and 4,394,448; see also non-U.S. patent documents WO8903849 and EP 0263740). Other methods for enhancing the efficiency ofoligonucleotide delivery are well known in the art and are alsocompatible with the present invention.

Having now described the invention, the same will be more readilyunderstood through reference to the following Examples which areprovided by way of illustration and which are not intended to limit thescope of the invention.

EXAMPLES Example 1 Cloning of Rat DRR-1

Isolation of cDNA Fragment.

Degenerate oligonucleotides were synthesized to highly conserved regionsof G-protein coupled receptors (transmembrane spanning domains 2 and 7)with the following nucleotide sequences:

5′ GG CCG TCG ACT TCA TCG TC(A/T) SEQ ID NO:15 (A/C)(T/C)C TI(G/T)CI(T/C) TIG C(A/C/G/T)G 3′ (TM2:sense); and 5′ (A/G)(C/A/T)(A/T) (A/G)CA(A/G)TA SEQ ID NO:16 IAT IAT IGG (A/G)TT 3′ (TM7:antisense).

Poly A+ mRNA was isolated from cultured fetal rat dorsal root ganglia(Sprague-Dawley). The mRNA was reverse transcribed using the FirstStrand cDNA Synthesis kit (Pharmacia Biotech), subjected to anamplification reaction by polymerase chain reaction (PCR) usingAmpli-Taq DNA (Perkin-Elmer Cetus) polymerase under the followingconditions: 3 minutes at 94° C., 40 cycles of 1 minute at 94° C., 45° C.and 72° C. A cDNA PCR fragment corresponding to approximately 650 bpswas isolated and subcloned in pGEM-T-vector (Promega Corporation). Thenucleotide sequence of the recombinant clone was determined using theT7-dideoxy chain termination sequencing kit (Pharmacia Biotech) and wasfound to be unique based upon searches of Genbank/EMBL databases.

The full length rat DRR-1 sequence was obtained from rat genomic DNAusing the 650 base pair fragment and the “Promoter Finder DNA Walkingkit” (Clontech, cat # K1806-1). This kit contains five libraries ofuncloned, adaptor-ligated genomic DNA fragments. The procedure involvestwo consecutive PCR reactions. Both reactions were done using the“Advantage Tth Polymerase Mix” also obtained from Clontech, followingthe conditions recommended by the vendor. The first PCR reaction wasperformed with the outer adaptor primer (AP1) provided in the kit and anouter, gene-specific primer (GSPI) derived from the sequence of theDRR-1 PCR fragment. The primary PCR mixtures were diluted and used as atemplate for the secondary (nested) PCR reaction with the nested adapterprimer (AP2) and a nested gene specific primer (GSP2). To obtain thesequence of the rat DRR-1 gene upstream of the sequence of the originalPCR fragment, the following oligonucleotides were used:

GSP1: oligo YF3B59-B, 5′-CGCAGATGAGGTAGTACAGCATCAC SEQ ID NO:17 GSP2:oligo MML-R1, 5′-CTGTGAGAGAGATGGTAACATACAG SEQ ID NO:18

From the first library, a fragment AP2-MMLR1 of 1.9 Kb was obtained andfrom the third library, a fragment of approximately 1.0 Kb was obtained.To identify the sequence downstream of the known sequence, the followingprimers were used:

GSP1: oligo YF3B59-F2, 5′-GCATCCTTGACTGGTTCTTCTCAG SEQ ID NO:19 GSP2:oligo MML-F1, 5′-GGGTGAGACTCATCATCATTTGTGG. SEQ ID NO:20

A fragment MMLF1-AP2 of approximately 1 Kb was obtained from the firstlibrary and a fragment of about 600 bp was obtained from the thirdlibrary. The composite sequence of 1154 nucleotides containing thecomplete predicted open reading frame of DRR-1 is shown in FIG. 1. Theopen reading frame codes for a 337 amino acid protein (FIG. 2) with apredicted molecular mass of 38.7 kD. The protein sequence contains allthe characteristic features of G protein-coupled receptors: sevenhydrophobic helices likely to represent transmembrane domains, potentialglycosylation site at the N-terminal extracellular domain (position 30)and a conserved NPXXY sequence at position 285–289.

Example 2 Cloning of Human DRR Receptor Genes

Two approaches were used to identify and clone novel human DNA sequenceshomologous and/or related to the rat DRR-1 gene. First, a human genomiclibrary was screened in the lambda vector, Fix II, (Stratagene Cat.#946203). Approximately 106 human genomic clones were plated andtransferred onto nitrocellulose membranes for hybridization with thefull length, 32P labeled, rat DRR-1 sequence as a probe. Thehybridization was performed at 42° C., overnight. The filters werewashed at room temperature at low stringency (1×SSC/0.1% SDS) to allowdetection of related but not necessarily identical sequences.

The inserted human DNA present in positive phages was amplified by PCRusing the “Expand PCR kit” from Boehringer-Mannheim under conditionsallowing accurate amplification of very large fragments of DNA. Theselong fragments of DNA were digested with various restriction enzymes andsubcloned into a plasmid vector. The portions of these clones whichhybridized with the rat DRR-1 gene probe were sequenced using the ABIcycle sequencing kit.

A second approach to identifying novel human sequences related to DRR-1involved the use of the polymerase chain reaction (PCR), performed ontotal human genomic DNA. Primers were synthesized based upon the humangenomic clones described above and were as follows:

HML.H, 5′-GCAAGCTTTCTGAGCATGGATCCAACCGTC, SEQ ID 21 and HML.Bg,5′-CCCTCAGATCTCCAATTTGCTTCCCGACAG,. SEQ ID NO:22

Amplification resulted in a fragments of approximately 1 kilobasecontaining the entire coding sequence of the human genes. Thesefragments obtained were subcloned into the pGEM-T (Promega) vector forDNA sequencing analysis.

Using the above strategies, six human clones were isolated:

-   clone 7, SEQ ID numbers 3 and 4;-   clone 18, SEQ ID numbers 5 and 6;-   clone 23, SEQ ID numbers 7 and 8;-   clone 24, SEQ ID numbers 9 and 10;-   clone 36, SEQ ID numbers 11 and 12;and-   clone 40, SEQ ID numbers 13 and 14.

None of these clones contain introns and their alignment may be seen inFIG. 3.

At the amino acid sequence level, the rat DRR-1 clone is 47% to 49%identical to the human clones.

At the nucleic acid level, the rat DRR-1 clone is 56% to 58% identicalto the human clones. The level of sequence identity within the humanclones (7, 18, 23, 24, 36, 40) is very high, between 77% and 98% at theamino acid sequence level. All the human sequences were used as queriesto search for homologies in public databases (Genbank, Swissprot, EST).No identical sequences were detected. The closest matches were tomembers of the mas oncogene family of proteins. The overall amino acidsequence homology between rat DRR-1 and any of the isolated human genesvaried from 47 to 50%. However some stretches display a much higherlevel of sequence homology, particularly the regions encoding theputative transmembrane domain III and VII (TM3 and TM7) and theintracellular loops 2 and 3 where the homology between the rat sequenceand its human homologue is around 80%.

Example 3 In Situ Hybridization Experiments

Preparation of Tissue: Adult male Sprague-Dawley rats (˜300 gm; CharlesRiver, St-Constant, Quebec) were sacrificed by decapitation. Brain andspinal cord with dorsal root ganglia attached were removed, snap-frozenin isopentane at −40° C. for 20 s and stored at −80° C. Frozen humanbrain, spinal cord and dorsal root ganglia were obtained from the Brainand Tissue Bank for Developmental Disorders, University of Maryland atBaltimore, according to the strictest ethical guidelines. Frozen tissuewas sectioned at 14 m in a Microm HM 500 M cryostat (Germany) andthaw-mounted onto ProbeOn Plus slides (Fisher Scientific, Montreal,Quebec). Sections were stored at −80° C. prior to in situ hybridization.

Synthesis of Riboprobes: The plasmid pGemT-3b32 GPCR was linearizedusing either SacII and Not 1 restriction enzymes. Sense and antisenseDRR riboprobes were transcribed in vitro using either T7 or SP6 RNApolymerases (Pharmacia Biotech), respectively in the presence of[35S]UTP (˜800 Ci/mmol; Amersham, Oakville, Ontario). The plasmidpGemT-Clone 36 GPCR was linearized using SacII and Pst 1 restrictionenzymes. Sense and antisense Clon36 riboprobes were transcribed in vitrousing either SP6 or T7 RNA polymerases (Pharmacia Biotech), respectivelyin the presence of [35S]UTP. Following transcription, the DNA templatewas digested with DNAse I (Pharmacia). Riboprobes were purified byphenol/chloroform/isoamyl alcohol extraction and precipitated in 70%ethanol containing ammonium acetate and tRNA. Quality of labeledriboprobes was verified by polyacrylamide-urea gel electrophoresis.

In situ Hybridization: Sections were postfixed in 4% paraformaldehyde(BDH, Poole, England) in 0.1 M phosphate buffer (pH 7.4) for 10 min atroom temperature (RT) and rinsed in three changes of 2× standard sodiumcitrate buffer (SSC; 0.15 M NaCl. 0.015 M sodium citrate, pH 7.0).Sections were then equilibrated in 0.1 M triethanolamine, treated with0.25% acetic anhydride in triethanolamine, rinsed in 2×SSC anddehydrated in an ethanol series (50–100%). Hybridization was performedin a buffer containing 75% formamide (Sigma, St-Louis, Mo.), 600 mMNaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 1× Denhardt's solution (Sigma), 50(g/ml denatured salmon sperm DNA (Sigma), 50 (g/ml yeast tRNA (Sigma),10% dextran sulfate (Sigma), 20 mM dithiothreitol and [35S]UTP-labeledcRNA probes (10×106 cpm/ml) at 55° C. for 18 h in humidified chambers.Following hybridization, slides were rinsed in 2×SSC at RT, treated with20 (g/ml RNase IA (Pharmacia) in RNase buffer (10 mM Tris, 500 mM NaCl,1 mM EDTA, pH 7.5) for 45 min at RT and washed to a final stringency of0.1×SSC at 65° C. Sections were then dehydrated and exposed to KodakBiomax MR film for 21 days and/or dipped in Kodak NTB2 emulsion diluted1:1 with distilled water and exposed for 4–6 weeks at 4° C. prior todevelopment and counterstaining with cresyl violet acetate (Sigma).

Results: Of all regions examined within the neuraxis of the rat, DRR-1mRNA was exclusively expressed in dorsal root ganglia. High resolutionemulsion autoradiography showed accumulations of silver grainsexclusively over small and some medium size neurons. This unique andhighly restricted distribution pattern for DRR-1 was confirmed in therat embryo. Sagittal section of an E17 rat fetus showed that DRR-1 mRNAis confined to DRGs. All other structures of the rat embryo were devoidof any specific hybridization signal reinforcing the highly selectivenature of DRR-1 expression

The expression of human Clone 36 receptor was present in human fetaldorsal root ganglia but not in spinal cord. Specific hybridizationsignal for Clone 36 was not detected in any of the human adult CNStissues examined thus far. These include spinal cord, cortex,hippocampus, thalamus, substantia-nigra and periaqueductal gray (datanot shown). Presence of Clone 36 mRNA in adult DRGs remains to beexamined. Standard controls in which additional spinal cord with DRGsections were hybridized with rat DRR-1 antisense or Clone 36 sense35S-labeled probes displayed no specific hybridization signal.

Example 4 Northern Blots

Commercial rat and human multiple Northern blots containing 2 g of polyARNA from various tissues (Clontech) were used to determine theexpression and distribution of the rat DRR-1 message and its humanhomologues. Radioactively labeled probes were prepared as follows:twenty five ng of a 650 bp 3b-32 PCR fragment derived from rat DRR-1(see Example 1) or human clone 36 were random-prime labeled using theReady-to-Go DNA labeling kit (Pharmacia Biotech) and [32P]CTP (3000Ci/mmol/Amersham). The blot was prehybridized for 1 hour at 68° C. usingExpresshyb (Clontech) followed by hybridization (2×106 cpm/ml of probe)for one hour using the same conditions. Blots were washed at roomtemperature in 2×SSC, 0.05% SDS for 30 min. followed by 3× washes in0.2×SSC, 1% SDS at 50° C. for 60 min. and exposed at −80° C. to KodakBiomax film for 6 days.

Expression and Distribution of rat DRR-1: All the rat tissues studied(heart, brain, spleen, lung, skeletal muscle, kidney and testis) werenegative for the expression of DRR-1 following 2 weeks exposure whereasrat genomic Southern analysis revealed a 1.1 kb band when probed withthe same cDNA fragment.

Expression and Distribution of Human Clone 36: Northern blots containingRNA from various human tissues were probed with a radio-labeled DNAfragment from clone 36. All the human tissues studied (human fetalbrain, lung, liver and kidney and adult human cerebellum, cerebralcortex, medulla, occipital pole, frontal lobe, temporal lobe, putamen,spinal cord, amygdala, caudate nucleus, corpus callosum, hippocampus,total brain, subthalamic nucleus and thalamus) were negative for theexpression of this receptor following 2 weeks exposure.

Example 5 Calcium Signaling in Response to Angiotensin I–III

The coding sequence of human clone 24 was transferred into a pcDNA3vector and modified to add a haemaglutinin tag at the C-terminus of thereceptor sequence. This clone, designated as pcDNA3-HML-HA24 wastransfected into HEK293 cells using a modified CaCl₂ method (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989)). The cells were maintained in culture medium at 37° C., 5%CO₂ and diluted 10 fold every 3 days.

The cells were inoculated in 90 mm tissue culture dishes (5×105 cellsper flask) in Dulbecco's Modified Essential Medium (DMEM, Gibco BRL),supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100μg/ml streptomycin and 0.25 μg/ml fungizone. One day after inoculation,cells were transiently transfected with 30 μg of plasmid DNA per dish.The cells were harvested 48 hours post transfection for analysis. Theexpression of the gene was first checked by immunoprecipitation andwestern blotting with an anti-haemaglutinin antibody. A protein ofapproximately 43 KD was detected in stably as well as transientlytransfected HEK293 cells, but not in control cells.

Stably transfected HEK293 cells were obtained after approximately 21days of selection in culture medium containing 800 μg/ml G418. Calciumsignaling measurement was performed with one of these stably transfectedcell line, 293/pcDNA3-HML-HA24. The cells were grown on a 24 mm roundglass cover slides to 50–70% confluence. After rinsing the cells with1.8 NBS buffer (135 mM NaCl, 5 mM KCl, 1.2 mM MgCl₂, 1.8 mM CaCl₂, 5 mMglucose and 10 mM HEPES, pH 7.3), the cells were incubated for one hourat room temperature in the presence of 0.5 ml of 3.5 μM FURA-2 AM(Molecular Probe, F-1221) diluted in 1.8 NBS. The cells were then rinsedthree times with 1.8 NBS and incubated for a further 30 minutes at roomtemperature. The calcium displacement was measured using a PTI (PhotonTechnology International) D104 photometer equipped with a PTI Delta RAMHigh speed multiwavelength illuminator, a PTI SC500 Shutter controller,a PTI LPS220 ARC lamp supply and the PTI FELIX software, v.1.2. Groupsof 2 to 8 cells were chosen and isolated with the photometer diaphragm.The cells were exposed to 340 and 380 nm light and the 510 nm lightemitted by the cells was recorded. Angiotensin I, II and III, were addedsuccessively—in various order from one experiment to the next—followedby bradykinin as a positive control. Upon stimulation with angiotensinII and angiotensin III, a significant response was obtained. Addition ofangiotensin I produced no response.

All references cited herein are fully incorporated by reference. Havingnow fully described the invention, it will be understood by one of skillin the art that the invention may be performed within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

1. A substantially pure protein comprising SEQ ID NO:3.
 2. The proteinof claim 1 wherein the protein consists of SEQ ID NO:3.
 3. A compositioncomprising the protein of claim 1.